Intracellular killing in D. discoideum: role of Vps13F and LrrkA

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Thesis

Reference

Intracellular killing in D. discoideum: role of Vps13F and LrrkA

BODINIER, Romain

Abstract

Intracellular killing is a complex process by which phagocytic cells eliminate microorganisms, once engulfed. In human tissues, intracellular killing is vital to fight off invading pathogens such as Klebsiella pneumoniae or for tissue homeostasis. Intracellular killing is mainly carried out within macrophages and neutrophils. Using Dictyostelium discoideum as a model organism for macrophages enables us to perform large scale random mutagenesis screen to find genes involved in intracellular killing of K. pneumoniae. Two of them have already been described: kil1 and kil2. Kil1 is a sulphotransferase and Kil2 a phagosomal magnesium pump.

We characterized two new genes involved in intracellular killing: vps13F and lrrkA. Vps13F is most likely involved in trafficking IC killing effectors to the phagosomes and LrrkA is at the convergence between sensing bacterial cue (i.e. folate) and activating intracellular killing mechanisms by respectively enhancing motility when folate is present and regulating Kil2 activity during the phagosome maturation.

BODINIER, Romain. Intracellular killing in D. discoideum: role of Vps13F and LrrkA . Thèse de doctorat : Univ. Genève, 2020, no. Sc. Vie 43

DOI : 10.13097/archive-ouverte/unige:143040 URN : urn:nbn:ch:unige-1430405

Available at:

http://archive-ouverte.unige.ch/unige:143040

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE FACULTÉ DE MEDECINE Section LIFE SCIENCES

Département de Physiologie Cellulaire et Métabolisme Professeur P. Cosson

Intracellular killing in D. discoideum: role of Vps13F and LrrkA.

THÈSE

présentée aux Facultés de médecine et des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences en sciences de la vie,

mention Sciences biomédicales

par

Romain Bodinier

de

Boulogne-billancourt (France)

Thèse N

^o

43 GENÈVE

Migros Printshop Plainpalais

2020

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Table of Contents

RÉSUMÉ ... 4

SUMMARY ... 5

ACKNOWLEDGEMENTS... 6

INTRODUCTION ... 7

I. DICTYOSTELIUM DISCOIDEUM A PROFESSIONAL PHAGOCYTE MODEL………8

II. PHAGOCYTOSIS: COMPARATIVE ANALYSIS IN HUMAN AND D. DISCOIDEUM………..10

Binding and recognition of particle ...11

Phagocytic cup: formation ...14

Phagocytic cup: guiding and closure ...15

III. PHAGOSOMAL MATURATION: COMPARATIVE ANALYSIS IN HUMAN AND D. DISCOIDEUM……….17

Rab GTPases regulates phagosome maturation ...17

The recycling machinery ...19

IV. INTRACELLULAR KILLING MECHANISM: COMPARATIVE ANALYSIS IN HUMAN AND D. DISCOIDEUM…………..23

Acidification of the phagosome lumen. ...23

ROS production in the phagosome. ...24

Lysosomal enzymes ...27

Lysozyme ...28

Antimicrobial peptides ...29

Nutritional immunity ...29

Metal poisoning ...31

Xenophagy ...33

Unexplored mechanisms of IC pathogen recognition and killing in D. discoideum...35

V. IC KILLING IN D. DISCOIDEUM: THE CASE OF K. PNEUMONIAE………...37

K. pneumoniae is heavily equipped to resist IC killing ...37

Role of Phg1A/Kil1 ...38

Role of Kil2 ...38

OBJECTIVES OF MY THESIS ... 40

MATERIALS AND METHODS ... 41

I. MEDIA, BUFFERS AND SOLUTIONS……….41

II. ANTIBODIES……….42

III. ANTIBIOTICS………42

IV. D. DISCOIDEUM CELL LINES………..42

V. BACTERIAL STRAINS………..43

VI. PLASMIDS………..43

VII. PRIMERS………43

VIII. CELL CULTURE……….44

IX. GENERATION OF KNOCK-OUT D. DISCOIDEUM CELLS………...45

X. DNA-RELATED PROTOCOLS………47

XI. PHAGOCYTOSIS ASSAY………51

XII. LIVE MICROSCOPY……….……..51

XIII. CELL FIXATION AND IMMUNOFLUORESCENCE……….………..57

RESULTS ... 58

I. VPS13F ALTERS INTRACELLULAR KILLING IN A KIL2-INDEPENDENT MANNER IN D. DISCOIDEUM…………58

Early characterization of Vps13F. ...58

Vps13F links bacterial recognition and intracellular killing in Dictyostelium ...59

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II. LRRKA ALTERS INTRACELLULAR KILLING IN A KIL2-DEPENDENT MANNER IN D. DISCOIDEUM………82

First characterization of LrrkA KO mutant ...82

LrrkA links folate sensing with Kil2 activity and intracellular killing...83

Manuscript: LrrkA IC killing supplementary data ...97

III. LRRKA IS A FOLATE-SENSITIVE MOTILITY SWITCH………101

LrrkA regulates multiple cellular functions. ... 101

LrrkA relays folate activation and controls cell motility and phagocytosis ... 102

Manuscript: LrrkA phagocytosis supplementary data ... 118

DISCUSSION ... 122

I. THE ADVANTAGES TO GO “LIVE"………122

A better understanding of K. pneumoniae Kil1/Kil2 IC killing pathways ... 122

Limitations of the new IC killing assay... 122

K. pneumoniae is presumably not killed directly by acid exposure. ... 123

Proteolysis in the maturing phagosome. ... 123

II. LRRKA IS INVOLVED IN SENSING, MOTILITY AND PHAGOCYTOSIS IN D. DISCOIDEUM………..124

Folate, an essential signal ... 124

LrrkA, a pivotal kinase in the folate-sensing pathway ... 124

LrrkA could integrate LPS and cAMP signals. ... 125

III. VPS13F EXHIBITS ONLY A SUBSET OF VPS13P FUNCTIONS………..125

IV. LRRKA MAY SHARE SEVERAL FUNCTIONS AS LRRK2………..126

REFERENCES ... 127

APPENDIXES ... 142

I. MANUSCRIPT:ROLE OF SPDA IN CELL SPREADING AND PHAGOCYTOSIS IN DICTYOSTELIUM……….142

II. MANUSCRIPT:SPDASUPPLEMENTARY DATA………160

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RÉSUMÉ

Contexte : l’élimination intracellulaire est un processus complexe par lequel des cellules phagocytaires éliminent des microorganismes après les avoir absorbés. Chez l’humain, l’élimination intracellulaire est tout aussi vitale pour combattre des agents pathogènes tels que Klebsiella pneumoniae que pour maintenir l’homéostasie des tissus. C’est le plus souvent au sein des macrophages et des neutrophiles que s’opère ce processus. Dictyostelium discoideum est régulièrement utilisé comme organisme modèle pour les macrophages, ce qui nous a permis de réaliser un criblage mutagénétique aléatoire à grande échelle dans le but d’identifier des gènes impliqués dans l’élimination intracellulaire de K. pneumoniae. Deux d’entre eux ont déjà été décrits : kil1 et kil2. Kil1 est une sulphotransférase et Kil2 est une pompe à magnésium phagosomale. Ces deux gènes ne sont ni dans la même voie métabolique, ni complémentaires. Qui plus est, notre connaissance des régulateurs et des effecteurs de ces voies métaboliques est lacunaire.

But : Identifier de nouveaux gènes potentiellement impliqués dans l’élimination intracellulaire au sein de D.

discoideum.

Méthode : À l’issue du criblage mutagénétique aléatoire, nous avons caractérisé les mutants présentant un défaut d’élimination intracellulaire de K. pneumoniae, ainsi que leurs liens avec kil1 et kil2. Cette caractérisation repose essentiellement sur des observations de cellules vivantes en microscopie à fluorescence.

Résultats : Nous avons trouvés deux gènes impliqués dans l’élimination intracellulaire : vps13F et lrrkA.

Vps13F est une protéine de la famille des vacuolar sorting protein et LrrkA de la famille des leucine rich repeats kinase. À la suite du développement de notre nouvelle analyse de l’élimination intracellulaire, nous avons mesuré le temps médian nécessaire pour qu’une cellule de la souche sauvage élimine une bactérie K.

pneumoniae. Ce temps médian est de 7.5 minutes. En comparaison avec la souche sauvage, les cellules mutantes vps13F KO et lrrkA KO mettent respectivement 18 minutes et 25 minutes. Néanmoins, elles ne présentent pratiquement aucunes anomalies durant leur croissance, leur développement, ni dans leur voie endocytique.

LrrkA et Vps13F ne sont pas situées dans la même voie métabolique d’élimination intracellulaire. Comparé aux mutants simples, le double mutant Δvps13FΔkil2 présente un défaut additionnel d’élimination intracellulaire.

Ceci n’est pas le cas du double mutant ΔlrrkAΔkil2, dont le défaut peut, à l’instar du mutant kil2, être partiellement corrigé par l’ajout de Mg²⁺.

Contrairement à nos attentes, nous avons non seulement découvert que l’ajout de folate peut stimuler l’élimination intracellulaire, mais aussi que celle-ci dépend de la voie métabolique affectée par les mutants.

L’ajout extracellulaire de folate ne stimule l’élimination intracellulaire, dans le cas des mutants simples lrrkA KO et vps13F KO, que le mutant vps13F. Pour confirmer l’hypothèse que la reconnaissance du folate est critique pour une élimination intracellulaire efficace, nous avons montrés que les mutants insensibles au folate, far1 KO and fspA KO, présentent eux aussi un défaut d’élimination intracellulaire.

De surcroît, l’addition de folate ne stimule pas la mobilité du mutant lrrkA, ce dernier étant constamment plus mobile que la souche sauvage. Ce phénotype induit par ailleurs une phagocytose plus importance chez le mutant que pour la souche sauvage.

Conclusion : Vps13F est certainement impliqué dans le trafic vers le phagosome des effecteurs de l’élimination intracellulaire. Nos prédictions génétiques supportent plus précisément que ce sont les enzymes lysosomal sulfatées par Kil1 qui sont concernées.

LrrkA, de son côté, est à la convergence de la détection de signal bactérien (i.e. folate) et de l’activation des mécanismes d’élimination intracellulaire. Cette dernière passe par l’activation de la motilité en présence de folate et de la régulation de l’activité de Kil2 durant la maturation du phagosome.

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SUMMARY

Background: Intracellular killing is a complex process by which phagocytic cells eliminate microorganisms, once engulfed. In human tissues, intracellular killing is vital to fight off invading pathogens such as Klebsiella pneumoniae or for tissue homeostasis. Intracellular killing is mainly carried out within macrophages and neutrophils. Using Dictyostelium discoideum as a model organism for macrophages enables us to perform large scale random mutagenesis screen to find genes involved in intracellular killing of K. pneumoniae. Two of them have already been described: kil1 and kil2. Kil1 is a sulphotransferase and Kil2 a phagosomal magnesium pump.

Both genes are involved in intracellular killing pathways that do not complement each other’s phenotype.

Furthermore, our knowledge of regulators and effectors in these pathways is sparse.

Aim : Increase the number of candidate genes implicated in intracellular killing in D. discoideum.

Method: Characterize mutants from a random mutagenesis screen for intracellular killing deficient mutants of K. pneumoniae, as well as their mutation dependencies on kil1 or kil2. We focused on fluorescence based live cell imaging techniques.

Results: We found two genes implicated in IC killing: vps13F and lrrkA. Vps13F is a cytosolic protein from the vacuolar protein sorting family, and LrrkA is a cytosolic kinase from the Leucine Rich Repeats kinase family. Following the development of a new intracellular killing assay, we measured the median time for WT D. discoideum cells to kill a single K. pneumoniae at 7.5 min. Compared to the WT, vps13F KO cells are at 18min and lrrkA KO cells at 25min. vps13F and lrrkA mutants nevertheless display virtually no abnormalities during growth, development, or in their endocytic pathway.

LrrkA and Vps13F work in separate IC killing pathways. Compared to single mutants, the double mutant Δvps13FΔkil2 exhibits an additive IC killing defect, whereas ΔlrrkAΔkil2 does not. In addition, lrrkA KO cells IC killing defect can be reversed by adding Mg²⁺, a phenotype observed in kil2 KO cells.

Unexpectedly, folate can stimulate IC killing. This stimulation is pathway-dependent, as exogenous addition of folate boosts IC killing in vps13F KO but not in lrrkA KO. This unexpected folate-dependent IC killing stimulation result is reinforced by both folate-sensing deficient mutants, fspA and far1 KO cells, being IC killing deficient as well.

Additionally, lrrkA KO cells are insensitive to the stimulation of motility by folate and are constantly more motile than the WT, resulting in increased phagocytosis compared to WT.

Conclusion: Vps13F is most likely involved in trafficking IC killing effectors to the phagosomes. Genetic prediction suggests more specifically a role in trafficking Kil1-sulfated lysosomal enzymes.

LrrkA is at the convergence between sensing bacterial cue (i.e. folate) and activating intracellular killing mechanisms by respectively enhancing motility when folate is present and regulating Kil2 activity during the phagosome maturation.

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ACKNOWLEDGEMENTS

« Ce n'est pas que la vie soit courte, c'est que le temps passe vite... » et sur ces paroles d’Henry Jeanson, je remercie mes parents pour avoir non seulement rempli la première condition nécessaire au travail de thèse, c’est-à-dire l’existence, mais aussi de continuer à accompagner avec amour depuis toujours.

La deuxième condition est d’avoir un directeur de thèse et cette dernière c’est toi, Pierre, qui l’a remplie. Pour cela je ne sais comment te remercier. Le dernier conseil de mon maître de thèse était de choisir un professeur avant de choisir un sujet de thèse, et je le remercie pour ce conseil. Merci pour ton accompagnement et tes encouragements tout au long de ces 5 années.

Il y a peu de mots pour décrire tous les sentiments d’affection et même d’amour que j’ai pour l’équipe du laboratoire. Jade, Ayman, Alex, Jackie, Philippe, Tania, Estelle, Chéryl, Otmane, Cyril et Xénia, vous avez été formidables. Tout comme ce comptoir à l’entrée de nos bureaux, lieu de vie, de rencontres, de fêtes, de débuts de discussions, et de soutien dans les instants de faible glycémie ; ainsi que cette porte, ornée de nos souvenirs, et citations.

L’équipe ne saurait être au complet si je ne remerciais pas Anna, Astrid, c’était un plaisir de travailler dans un laboratoire aussi bien tenu.

Un mot aussi pour Thierry Soldati et François Letourneur qui, je l’espère, trouveront dans mes publications des pistes et réponses pour continuer de faire avancer leur recherche. Merci encore pour votre temps, et pour avoir le courage de lire cette thèse.

La vie au laboratoire ne s’arrêtait pas aux murs de celui de Pierre. Si je remercie les comités qui ont organisé les PhD retreats, c’est surtout Semia et Daniele qui pour moi ont été deux amis en dehors du labo. Je profite de l’occasion aussi pour remercier l’équipe de « ma thèse en 180 secondes » qui m’a aidé à me révéler un goût pour la vulgarisation scientifique.

Merci aussi à Karina, Margaux, Laure, Siroune et Marion pour leur soutien.

Pour finir je souhaite remercier les associations Geneva E-Sport et Swiss Esports Federation. Des heures investies avec le sourire et dont a jailli des amitiés, des projets, des aventures… Je recommande à tous les doctorants d’avoir une passion comme celle-ci qui se concrétise sur le côté. La thèse est un marathon, mais ayant été aussi bien entouré, je suis heureux du chemin que nous avons fait ensemble.

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INTRODUCTION

Humans and bacteria peacefully cohabit most of the time (Turnbaugh et al., 2007). Indeed, numerous bacteria in our gut or on our skin are beneficial to human health. Only few bacteria have developed virulent strategies that harm the human body. In addition, the human body is equipped with an immune system preventing undesirable microorganism colonization of tissues (Tosi et al., 2005).

The first line of defense of the immune system is called the innate immune system. It notably comprises a set of cells that exhibit microbicidal activities, allowing them to contain or eradicate invading microorganisms (Silva et al., 2012). Their modus operandi is the following: they track microorganisms, ingest them in a membrane-enclosed organelle called a phagosome, and eventually kill and digest them. Uptake of particles larger than 200nm is called phagocytosis. Immune cells with remarkably high phagocytic activity are referred to as professional phagocytes and include macrophages, neutrophils, dendritic cells, osteoclasts, and eosinophils (Gordon et al., 2016). In contrast cells having a low phagocytic activity are designated as nonprofessional phagocytes and include fibroblasts, epithelial cells, and endothelial cells. Although nonprofessional phagocytes usually do not ingest microorganisms, they play a role in the phagocytic ingestion and elimination of apoptotic bodies (Gordon et al., 2016).

Once a foreign microorganism is sequestered in the lumen of a phagosome, a series of complex phagosomal maturation events occur, allowing intracellular killing and degradation of the microorganism. Within minutes after closure, the phagosome becomes a highly acidic, degradative and oxidative compartment (Pauwels et al., 2017). An extensive literature on human professional phagocytes describes their intracellular microbicidal activities, yet it remains unclear which of these mechanisms are most necessary for intracellular killing and if we have today discovered the whole spectrum of mechanisms. It is also largely unclear whether killing of different microorganisms relies on similar or distinct killing mechanisms.

Phagocytosis of microorganisms plays a second role in the immune system as proper degradation of invading microorganisms is required to efficiently trigger the immune system’s second line of defense: the adaptative immunity. Unlike innate immune cells that use a defined set of receptors to recognize foreign molecules and pathogens, cells of the adaptive immune system respond to foreign antigen fragments displayed by the major histocompatibility complex (MHC) class I and II at the surface of macrophages, dendritic cells, and to a lesser extent neutrophils (Vono et al., 2017). Therefore, efficient digestion of foreign particles in the phagosome is necessary to properly load foreign antigens fragments on the MHCs (Litman et al., 2010).

In summary, studying intracellular killing mechanisms within the phagosome of professional phagocytic cells is essential to understand how the human body protects itself against foreign microorganisms.

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INTRODUCTION

I. Dictyostelium Discoideum a professional phagocyte model

D. discoideum is classified as an amoeba belonging to the phylum Amoebozoa, infraphylum Mycetozoa.

Historically, amoebae were first observed in 1755 by Rösel von Rosenhof and described as small motile protozoon. In 1822 the name Amiba, from the Greek amoibè (ἀμοιβή) meaning "change", was coined by Bory de Saint-Vincent and last changed to Amoeba 10 years later by Ehrenberg. In 1869, Brefeld observed Dictyostelids, a family of amoeba, and Raper in 1935 performed the first characterization of a prominent member of the family: D. discoideum.

D. discoideum, originally isolated from decaying leaves from a hardwood forest in North Carolina mountains, can be found in two main states: a vegetative state characterized by motile single cells of approximately 5-10µm diameter capable of feeding on a bacterial lawn ; on the opposite, the developmental stage corresponds to a multicellular development cycle where starving cells aggregate to form a slug, which undergoes morphogenesis into a fruiting body (Raper, 1935) (Fig.1).

The capacity of D. discoideum amoebae to undergo a complex multicellular developmental cycle is a remarkable feat. Starving cells release cAMP as a signal to regroup. When 10⁵to 10⁶cells aggregate, a slug is formed.

Formation of the slug induces a complete transcriptional change for most of the cells. This change triggers tropism toward light, heat, and humidity enabling the slug to migrate towards favourable grounds. The last step includes differentiation into two distinct subpopulations of cells in the fruiting body: the stalk and the sorus/spores (Katz, 2002). Eventually, the spores germinate and give rise to new vegetative cells. This facultative multicellular process is the reason why D. discoideum is often nicknamed a “social amoeba”.

Fig.1 D. discoideum life cycle

(A) Amoeboid cells feed on bacteria and replicate by binary fission. (B) The development cycle is initiated upon resource depletion, and aggregation occurs when starving cells secrete cAMP. (C) The aggregating cells organize to form the mound stage, enclosed within an extracellular matrix and (D) continue to develop into the standing slug. (E) Depending on its environment, the standing slug either falls over to become a migrating slug that moves towards heat and light or (F) proceeds directly to the culmination stages that (G) ultimately produce the fruiting body, which consists of a spore- containing structure, the sorus, and a stalk of dead cells. (H) Spores are released from the sorus and germinate into

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INTRODUCTION

D. discoideum was used as a model organism for studying phagocytosis and intracellular (IC) killing of bacteria in 1978 by Depraitère (Depraitère and Darmon, 1978), and it was used as a model for studying phagocytosis in the following decades. Hägele opened the path to using D. discoideum as a model phagocytic cell exposed to pathogenic bacteria in a study of Legionella in 2000 (Hägele et al., 2000). D. discoideum infection by Legionella recapitulates the same pathogenicity as in macrophages, leading the authors to hypothesize that amoebae are probably the original target of Legionella in the environment. In 2005 the sequencing of the full genome of D.

discoideum was published and made available on dictybase (Eichinger et al., 2005) revealing that most of the genes involved in phagocytosis, phagosome maturation, and IC killing are strikingly conserved from D.

discoideum to human (Boulais et al., 2010). The conservation of these genes is one of the main reasons to use D. discoideum as a model phagocytic cell (Dunn et al., 2018) and allowed D. discoideum to be used as a model organism for studying the infection course of a dozen pathogens (Table 1).

Bacterial pathogens References

Legionella pneumophila Hägele et al., 2000

Mycobacterium avium, M. marinum, M.

tuberculosis

Skriwan et al., 2002, Hagedorn et al., 2007, Solomon et al., 2003

Pseudomonas aeruginosa Cosson et al., 2002

Vibrio cholerae Pukatzki et al., 2006

Klebsiella pneumoniae Benghezal et al., 2006

Neisseria meningitidis Colucci et al., 2008

Burkholderia cenocepacia Aubert et al., 2008

Salmonella enterica/typhimurium Jia et al., 2009

Francisella noatunensis Lampe et al., 2016

Although the mouse is a standard model organism for studying professional phagocytic cells, and more generally the immune system in mammals, it remains challenging to perform large scale systematic analysis of gene products directly implicated in IC killing. (Swearengen, 2018). Using D. discoideum for large random mutagenic screens and subsequent characterization of mutants presents five experimental advantages: it is simple, short, scalable, cheap and robust. Simple, because in optimal growth conditions, D. discoideum is in a vegetative state, which is characterized by freely moving single cells. Phenotypes observed are therefore mostly cell-autonomous reducing the complexity of the interpretations. Short, because D. discoideum doubling time is 6 hours. It is almost 5 times shorter than a standard macrophage cell line (Van Furth et al., 1987). Scalable, because D. discoideum cells grow in less stringent growth conditions to higher concentration. Cells grows at 20-25 C^o without the need to control for 02 and C02 . Cheap, because cells can be cultivated on a bacterial lawn or even in a cheap nutritive medium for axenic strains and D. discoideum cells do not required treated culture plastic to grow. Robust, because D. discoideum genetic modifications are better controlled and defined compared to the diploid mouse macrophage genome. D. discoideum cells have a haploid genome and strategies to generate libraries of KOs or KIs mutants are readily available. Recent adaptation of the Crisper-Cas9 system to D. discoideum facilitates even more the generation of mutants (Sekine et al., 2018).

In numerous laboratories, D. discoideum strains used are called axenic, meaning they can grow on liquid media without bacteria. In a rich medium, cells remain in a vegetative state with high phagocytic and macropinocytic rate avoiding entry in the multicellular stage. In 2015, Bloomfield et al. showed that the axenic phenotype is due to the loss of the RasGAP: NF1. Excessive Ras activity, generated by NF1 loss, increases macropinocytic activity enabling the cells to survive in nutrient-rich media (Bloomfield et al., 2015). It does not affect axenic cells capacity to grow on a bacterial lawn and axenic cells are still considered a valid model for macrophages.

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INTRODUCTION

II. Phagocytosis: comparative analysis in human and D. discoideum

Phagocytosis comes from Ancient Greek phagein (φαγεῖν) and kytos (κύτος) meaning “to eat” and “cell”. It is the process allowing cells to engulf particles larger than 200 nm. The term was coined by Carl Friedrich Wilhelm Claus to describe the set of cells capable of absorbing and degrading citrus thorns inserted into starfish larvae observed in 1882 by ÉlieMechnikov.

In human cells, phagocytosis role extends beyond immunity, for example it prevents severe anemia by enabling clearance of erythroblast nuclei (Kawane et al., 2001), it prevents toxic iron deposits in the kidney by removing senescent erythrocytes (Theurl et al., 2016), it enables correct synaptogenesis by pruning the synapses that are superfluous or inactive to optimize neuronal communication (Petanjek et al., 2011) and prevents permanent brain damage by clearing cellular debris during brain ischemia (Morizawa et al., 2017), it clears the lumen of seminiferous tubules from apoptotic cells for efficiency spermatogenesis (Shaha et al., 2010), and it even prevents blindness by clearing the shed outer segments of photoreceptor cells in the retina (Nandrot et al., 2000). Underlying this plethora of roles, we still find common cellular mechanisms.

At the cellular level, phagocytosis is a three step process: first, receptors present at the cell surface recognize the foreign particle; second, a local remodeling of the cytoskeleton and plasma membrane is induced by a signaling cascade, and forms the phagocytic cup around the foreign particle; third, the phagocytic cup closes and forms an intracellular compartment called a phagosome (Desjardins et al., 2005) (Fig.2).

Fig.2 Phagocytosis – General overview

Phagocytosis is a three step process: first, receptors present at the cell surface recognize the foreign particle (in orange);

second, a local remodeling of the cytoskeleton and plasma membrane is induced by a signaling cascade, and forms the phagocytic cup around the foreign particle; third, the phagocytic cup closes and forms an intracellular compartment called a phagosome,

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INTRODUCTION

In D. discoideum, phagocytosis, like for numerous unicellular organisms, is mainly used for feeding purposes.

D. discoideum cells mainly phagocytose bacteria, and the same general steps are conserved. Only internalization of D. discoideum lectin (Discoidin I) coated bacteria differs from the typical course of phagocytosis and phagosome maturation. Discoidin-coating of extracellular bacteria protects them from IC killing (Dinh et al., 2018). When fluorescent Discoidin-coated E. coli were fed to D. discoideum, the fluorescent signal within the cells persisted on average 25 min, whereas it persisted 4 min with fluorescent non-coated E. coli. The results indicated that E. coli survived longer within the phagosomes when coated by Discoidin I. The authors hypothesized that Lectin-induced modified bacterial internalization can be distinguished from phagocytosis and has a different maturation process. One explanation could be that during starvation, it might be beneficial to store bacteria as food and delay their IC killing, to provide germinating spores with a readily available food source.

Binding and recognition of particle

Human phagocytic cells exhibit at the plasma membrane, endosomal membranes and in the cytosol, specific pattern recognition receptors (PRR). These PRRs recognize either damage-associated molecular pattern (DAMP) molecules or microbial-associated molecular pattern (MAMP) molecules (Tang et al., 2012) (Fig.3).

More specifically MAMPs ranges from components of the peptidoglycan cell wall to nucleic acids. Four types of PRRs directly bind them: toll-like receptors (TLRs), integrins, scavenger receptors, C-type lectins (Iwasaki et al., 2015). Another set of receptors, called opsonic receptors, recognize host-derived opsonins, from the Greek opsōneîn “to prepare for eating”, bound to foreign microorganisms. Opsonic receptors include FcγR (antibodies), CR3 (complement), Integrin alpha-5/beta-1 receptor (fibronectin), and Integrin alpha-v/beta-3 and alpha-v/beta-5 receptors (lactadherin) (Liu et al., 2013, Freeman et al., 2014) (Fig.3). Heterologous expression of lectins, scavenger receptors, integrins and FcγR in non-phagocytic cells is sufficient to trigger phagocytosis;

TLRs on the other hand do not (Flannagan et al., 2012). Although TLRs do not directly trigger phagocytosis, they play two important roles. First, they stimulate phagocytosis: incubation of macrophages with the TLRs ligands increases bacterial phagocytosis and conversely presence of TLRs antagonists reduces the phagocytic rate of the macrophages (Doyle et al., 2004). Second, TLRs activation induces a signaling cascade triggering inflammation, another innate immune system mechanism. In short, upon binding to MAMPs, TLRs recruit adaptor molecules possessing a Toll–interleukin 1 receptor (TIR) domains such as MyD88, Mal, TRIF, TRAM, and SARM. In turn, TIR adaptors activation leads to stimulation of mitogen activated protein kinases (MAPKs) and NF-κB, eventually upregulating phagocytosis and inflammation-related genes (Cen et al., 2018). Not all plasma membrane located PRRs are direct phagocytic receptors, but all do trigger a signaling cascade leading directly or indirectly to phagocytosis.

In D. discoideum opsonin-independent phagocytosis has been described: five integrin-like protein (Sib family), and three scavenger-like receptors (Lmp family) have been identified and characterized (Cornillon et al., 2006, Sattler et al., 2018) (Fig.4). The Sib family, which comprises 5 members (SibA-E), shows similarity with integrin β. Among them, SibA and SibC are involved directly in adhesion to substrate, beads and bacteria.

Moreover, all Sib proteins interact directly, via their membrane-proximal NPxY motif, with TalinA, an actin- binding protein (Cornillon et al., 2006, Cornillon et al., 2008). Both features make SibA and SibC good phagocytic receptors candidates. However, we should take into consideration that TalinA is mostly found at the posterior cortex during migration, whereas a recent study would suggest that TalinB, the second homolog of Talin in D. discoideum, is restricted to the leading edge of migrating cells. Compared to TalinA, TalinB contains a I/LWEQ domain and a villin headpiece domain in its C-terminal actin-binding region which seem to play a role in both TalinA and B segregation (Tsujioka et al., 2019). Phagocytic receptors binding TalinB could be better phagocytic receptor candidates as phagocytosis compares to a directed migration over a surface. SibA and C binding affinity to TalinB have not been measured yet. Concerning the Lmp family, which comprises 3

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INTRODUCTION

members (LmpA-C), they are homologs of LIMP-2/CD36 scavenger receptors. LmpA and C are found in endosomes and lysosomes similar to LIMP-2, and LmpB is present at the cell surface like CD36 (Sattler et al., 2018). LmpA genetic inactivation induces a severe defect in phagocytosis for beads as well as Gram+ and Gram- bacteria, whereas LmpB inactivation specifically inhibits uptake of mycobacteria and B. subtilis. Most likely LmpA affects actin dynamic as lmpA KO cells have a massive decrease of motility, and LmpB regulates phagocytosis triggering as binding to bacteria is not affected (Sattler et al., 2018). LmpB is an interesting PRR candidate, specific for Gram+ bacteria, and LmpA play a more global role in phagocytosis, motility and even phagosome maturation, which is heavily perturbed in lmpA KO cells.

Fig.3 Phagocytosis initiation in macrophages

1) In the case of opsonin-dependent phagocytosis: antibodies (Ab) and C3bi, opsonizing the bacteria, are bound respectively by FcR and CR3. In Ab bound-state, the FcR is quickly phosphorylated, Syk recognizes the phosphorylated immunoreceptor tyrosine-based activation motifs.

Activation of Syk, further stimulates RAC, which in turn activates the actin-branching Arp 2/3 complex.

In the case of C3bi binding to CR3, activation of CR3 stimulates PI3K activity, leading to PIP2

formation with binds WASP relieving inhibition of Arp 2/3.

2) In the case of opsonin-independent phagocytosis: the MAMPs are directly recognized by the receptors. Both integrins and scavenger receptors stimulate Src kinases after binding. Src kinases activates Rho, which will release the WASP inhibition on Arp2/3 to allow actin-branching. The represented scavenger is class A member MARCO which binds bacterial CpG DNA.

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Interestingly in D. discoideum no C-type lectins, nor TLR enchased in the plasma membrane have been characterized so far (Fig.4). Concerning TLRs, two cytosolic proteins in D. discoideum contain toll/interleukin 1 receptor (TIR) domains: TirA and TirB. TIR domains are a hallmark of TLRs, but neither TirA nor TirB contain both TLR canonical domains: leucine-rich repeats (LRR) and transmembrane domains. Despite not displaying all the features of a TLR, TirA still plays a role in efficient IC killing: tirA mutant cells exposed to an avirulent strain of Legionella exhibit a growth defect (Chen et al., 2007).

D. discoideum cells, unlike in human cells, use a single G-protein coupled receptor (GPCR), fAR1, to induce phagocytosis following binding to folate and lipopolysaccharide (LPS) (Pan et al., 2016, Pan et al., 2018) (Fig.4). Folate and LPS are two classical MAMPS: Folate is secreted by many bacterial species such as K.

pneumoniae and is a strong chemoattractant for D. discoideum and LPS are a component on the bacterial outer membrane. In human cells, LPS binds TLR4 and folate binds folate receptors, which are cell surface glycosylphosphatidylinositol-anchored glycoproteins. In contrast, D. discoideum cells use a single receptor for the two MAMPS. In addition, fAR1 is also different from other chemoattractant GPCRs: it belongs to the class C GPCR family and exhibits a VFT extracellular domain for sensing multiple ligands (Pan et al., 2018). The main features of fAR1 is that it binds folate and LPS and also mediates engulfment of K. pneumoniae, as far1 KO cells exhibit a specific phagocytosis defect (Pan et al., 2016). fAR1 is the only known GPCR bridging chemotaxis and phagocytosis.

Fig.4 Phagocytosis initiation in Dictyostelium discoideum

Like in macrophages, MAMPs are directly recognized by their cognate receptors eventually triggering phagocytosis. SibA shares similarities with mammalian β integrin: notably an extracellular Von Willebrandt A domain, a glycine-rich transmembrane domain and a highly conserved cytosolic domain interacting with TalinA. LmpB is a scavenger receptor similar to the mammalian CD36 scavenger receptor. fAR1 is a GPCR, binding folate and LPS directly inducing phagocytosis.

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Phagocytic cup: formation

The membrane cytoskeleton of human phagocytes is very dynamic, even in its “resting” state before induction of phagocytosis. In FRAP experiments, locally photobleached GFP-tagged cytoskeleton components (actin, α- actinin, ezrin and myosins) are replaced by their fluorescent counterparts within seconds. Cytoskeleton core components have a turn-over of a few seconds (Fritzsche et al., 2013). The dynamic remodeling activity of the cytoskeleton relies on a competition between two polymerizing F-actin networks: an actin branching system relying on Arp2/3, and a mesh system relying on Rho/formin/myosin (Lomakin et al., 2015). For phagocytosis to happen, the balance needs to tilt towards Arp2/3 branched structures, as formin-polymerized actin immobilizes transmembrane proteins obstructing membrane ruffling and the diffusion of non-tethered membrane proteins and even lipids (Ostrowski et al., 2016).

Tilting the balance begins by activation of the Src-Family Kinase (SFK), after the binding of the microorganism to PRRs. Activation of the SFKs recruits locally Syk, which in turn phosphorylates adaptors, kinases, and lipases. The unstimulated Arp2/3 complex is weakly active and requires stimulation by nucleation promoting factors (NPFs) to branch a novel actin filament at a 70° angle from an existing one (Goley and Welch, 2006).

Members of the Wiskott-Aldrich syndrome protein (WASP) family are known to be major enhancers of the Arp2/3 complex activity (Politt and Insall, 2009). The WASP family contains two principal classes of proteins : WASPs and SCAR/WAVEs. Conformation change of WASPs proteins from inactive to active is triggered by binding to PIP2, a phospholipid, whereas the Scar/WAVE complex is activated by the Rho GTPase Rac. Once activated both classes of proteins enhance Arp2/3 branching activity (Fig.5).

Remodeling the cytoskeleton contributes to an increased diffusion of non-engaged PRRs, thus favoring the formation of novel focal points of adhesion to the target. Moreover, further activation of integrins via Rap GTPases, downstream of Syk activation, provides a linkage between the target and the actin cytoskeleton via talin, kindlin, vinculin, and other associated proteins. The integrins engaged and tethered to the actin cytoskeleton are in fact sufficient to form diffusional barriers for a majority of protein and even lipid rafts (Maxson et al., 2018). The diffusional barrier is necessary to delimits a perimeter for optimal phagocytosis.

Furthermore, if the target is too voluminous, presence of the diffusional barrier enables the maturation of the open tubular phagosomes while limiting a potential inflammation through the leakage of its content (Maxson et al., 2018).

In addition to the Integrin-based diffusion barrier, the protrusion is consolidated by Bin-Amphiphysin-Rvs167 (BAR) domain containing proteins. The BAR proteins are involved in stabilizing membrane curvature.

Additionally, they bind directly the WASP family proteins, including N-WASP and WAVE, which then activate the Arp2/3 complex (Hanawa-Suetsugu et al., 2019).

Concerning the phagocytosis machinery in D. discoideum, overall the protrusion extension mechanism is very similar to that used by mammalian cells. MAMPs binding to PRRs leads to the stimulation of the SCAR/WAVE complex by Rac homologs, notably Rac1. The SCAR/WAVE complex activates Arp2/3 leading to actin branching that drives the formation of the phagocytic cup (Rivero and Xiong, 2016) (Fig.6). Similarly, segregation of membrane proteins is visible in the phagocytic cup by a mechanism restricting lateral diffusion (Mercanti et al., 2006). Additionally, BAR proteins stabilize the phagocytic cup. Among the BAR proteins in D. discoideum, only one localizes specifically at the protrusive rim of the cup and regulates both Rac and Ras activity: RGBARG. RGBARG induces protrusion formation by activating RacG, whilst restricting expansion of the cup interior by locally inhibiting Ras activity (Buckley et al., 2019).

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Phagocytic cup: guiding and closure

In addition to PRRS, membrane lipids are actively involved in phagocytosis (Bohdanowicz et al., 2013).

Phosphoinositides (PIs) are phospholipids that can be phosphorylated at different positions (3, 4 or 5) of their inositol head group. PIP2 and PIP3 play essential functions in phagocytosis. PIP2, mostly localized to the inner plasma membrane leaflet and constitutes 1–2% of plasma membrane lipids. PIP2 recruits and binds actin- binding proteins driving local actin branching (Mu et al., 2018). Conversion of PIP2 to PIP3, by the action of the PI3 kinase at the tip of the protrusion recruits myosins which exert contractile activity and function as a purse string to facilitate phagosome closure (Ostrowski et al., 2016). Phagosome closure eventually needs dephosphorylation of PIP3 and disappearance of PIP2 mediated by respectively by OCRL and Inpp5B (Bohdanowicz et al., 2012).

Fig.5 Molecular mechanism of phagocytosis in macrophages

1) Pseudopod extension. The extension is driven by actin polymerization by the Arp 2/3 complex. Activation of the Arp 2/3 complex depends on both relieving WASP inhibition and activating the Scar/Wave complex. WASP inhibition is relieved by binding PIP2. Scar/Wave complex activation is induced by RAC. RAC is notably stimulated after the production of PIP3 which results from the phosphorylation of PIP2 by PI3K. Finally, membrane supply, to support the extension of the phagosome cup, are delivered via fusion in the actin free bottom of the phagocytic cup.

2) Phagosome closure. PIP3 and PIp2 are depleted at the tip of the pseudopod by OCRL and Inpp5B, reinstating WASP inhibition on Arp 2/3. OCRL is bound to the cytoskeleton by PADDL and the myosin located at the tip of the pseudopod generated the contractile force to close the phagosome.

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Additionally, PIP3 also recruits Rho GAPs that inactivate Rac1, terminating actin polymerization at the base of the phagocytic cup. Concomitant cleavage of PIP2, to DAG and IP3, by phospholipase C (PLC), also releases actin-binding proteins such as cofilin, WASp, and ezrin at the base of the phagocytic cup. The joint action of Rho GAPs and PLC, leading to the loss of actin at the base of the phagocytic cup, allows for fusion of vesicles with the plasma membrane (Niedergang and Chavrier, 2004). In a nutshell, PIP regulation is crucial to the protrusion formation, phagosome sealing and membrane fusion at the nascent phagosome base (Fig.5).

Interestingly PIP2 is sensitive to the membrane curvature. New evidence suggests that it could autonomously induce receptor-independent phagocytosis. Binding of a solid particle curves the membrane, which induces a local sorting of PIP2. PIP2 recruits and activate moesin in turn binding Syk and leading to receptor-independent phagocytosis (Mu et al., 2018). The authors hypothesized that this mechanism it may explain how phagocytes recognizes almost all variations of solid.

In D. discoideum, the role of phosphoinositides is also established. PIP2 accumulates near engaged PRRs and recruits NPFs and actin-binding proteins involved in membrane deformation in a similar fashion. PIP2 is also phosphorylated by PI3K and cleaved by PLC. The PIP2 decrease allows actin disassembly at the base of the phagocytic cup for vesicle fusion. Regarding phagosome closure, Dd5P4, the Dictyostelium homolog of human OCRL, dephosphorylates PIP3 into PIP2 which is an important step for the phagosome closure (Loovers et al., 2007). In conclusion, similar to mammalian phagocytic cells, PIP2 decrease allows actin disassembly and PIP3

dephosphorylation is also necessary for phagosome closure (Fig.6).

Phosphoinositides also plays a role in phagosome maturation in both human phagocytes and D. discoideum cells and will be subsequently described (Buckley et al., 2019, Luscher et al., 2019).

Fig.6 Molecular mechanism of phagocytosis in Dictyostelium discoideum

The phagocytic cup extension is, like in macrophages, driven by actin polymerization catalyzed by the Arp 2/3 complex. In a similar fashion activation of the Arp 2/3 complex requires to relieve WASP inhibition and promote the Scar/Wave complex activation. WASP inhibition is relieved by PIP2 binding. Scar/Wave complex activation is done by RAC1. RAC1 is stimulated after production of PIP3. PIP3 production is catalyzed by PI3K from PIP2. Membrane supply to support the extension are delivered via fusion in the actin free bottom of the phagocytic cup. At the tip of the pseudopod Dd5P4, the OCRL ortholog participates in reinstating WASP inhibition on Arp 2/3. Myosin located at the tip of the pseudopod generates the contractile for to close the phagosome. MyoVII is probably the main driving force.

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III. Phagosomal maturation: comparative analysis in human and D. discoideum

The nascent phagosome quickly undergoes maturation. Within minutes after closure the phagosome becomes a highly acidic, degradative and oxidative compartment (Pauwels et al., 2017) (Fig.7). The maturation of the phagosome, both in human cells and in D. discoideum, is divided in three parts: fusion with the early endosomes, followed by fusion with late endosomes and finally with lysosomes. These three steps are a prerequisite for the phagosome to obtain its full microbicidal capacity. Interestingly, new material delivered to the phagosomes does not increase significantly the phagosome size nor decrease the size of the endosomes. This observation led to a model, coined “kiss and run” (Duclos et al., 2000), that favors repeated transient fusion events over a full fusion between compartments, although both events occur. The phagosomal maturation is also supported by a recycling machinery for protein sorting and recycling in order to allow several cycles of phagocytosis.

Rab GTPases regulates phagosome maturation

In human cells, the sequence of phagosome fusion with compartments of the endocytic pathway, as well as recycling of components from the phagosome, is highly regulated by Rab GTPases (Prashar et al., 2017) (Fig.7).

Rabs are small GTPases, that cycle between a GTP-bound active state to a GDP-bound inactive state. Rabs show homology to YPT1/SEC4, which regulates membrane trafficking between internal organelles in yeast.

Rabs are present on various organelles along the endocytic pathway and are often used to define the organelle identity (Gutierrez, 2013). Functionally, Rabs in conjunction with Rab adaptors and effectors, allow coordinated and sequential fusion of the phagosomes with vesicles (Gutierrez, 2013).

Over 20 Rabs are associated with phagosomes (Gutierrez, 2013). Among them, Rab5 and Rab7 where the first described and are essential for proper phagosome maturation (Chavrier et al., 1990). The first step of maturation is regulated by Rab5. Rab5 is recruited to phagosomes in a Rabex-5/Rabaptin-5 dependent manner (Horiuchi et al., 1997). Once Rab5 is recruited, it activates the Class III phosphoinositide3-kinase vacuolar protein-sorting 34 (VPS-34) (Kinchen et al., 2008). VPS-34 catalyzes the production of PI(3)P, which recruits proteins with PX or FYVE domains such as EEA1 (early endosomal antigen 1), PIKfyve, p40 (subunit of the NADPH oxidase) Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate), and the class C core vacuole/endosome tether (CORVET) complex (Vieira et al., 2004, Kinchen et al., 2008, Prashar et al., 2017).

At this step the phagosome is able to fuse with early endosomes, in an EEA1 and SNARE dependent manner (Christoforidis et al., 1999), and to transition to the second step. The transition from an early phagosome to a late phagosome is marked by the conversion from Rab5 to Rab7 and the CORVET to HOPS (homotypic fusion and vacuole-sorting) complex switch (Gordon et al., 2016). Both conversions depend on the Mon1–Ccz1 complex activity. Mon1 inactivates RAB-5 by displacing RABX-5 and Ccz1 recruits Rab7 (Nordmann et al., 2010). Following the Rab5-to-Rab7 switch, late phagosomes are devoid of Rab5 and Rab5-bound-CORVET complex, while decorated with active Rab7 and the Rab7-bound-HOPS complex. The HOPS complex activity is necessary for the fusion with late endosomes and Rab7 activity is a prerequisite for the centripetal movement of phagosomes. During maturation, phagosomes migrate from the cell periphery to the vicinity of the microtubule-organizing center (MTOC) where lysosomes are numerous (Harrison et al., 2003). Binding to the microtubule is carried by the Rab7 effector RILP and the cholesterol sensor ORP1L working together to recruit the dynactin complex (Li et al., 2016). The marker for the completion of phagosome to lysosome fusion are LAMP1 and 2 (Huynh et al., 2007). Once acquired the phagosome can fuse efficiently with lysosomes in a SNAREs dependent fashion. A process again partly under the regulation of PIPs, where PI(3)P is required to prime SNAREs for fusion and PI(4)P is required during and after tethering of the HOPS complex to lysosomes (Jeschke and Haas, 2017).

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Fig.7 Maturation of the phagosome

Once the phagosome is sealed, the phagosome matures during a successive series of transient fusions with endosomes and lysosomes, leading to the formation of the phagolysosome. The first maturation marker, Rab5, is recruited to the phagosome by the Rabex/Rabaptin complex delivered via fusion with early endosomes.

Rab5 activates VPS34 which produces PI(3)P. PI(3)P production serves to dock protein with PIP3-binding FYVE domains (PykeFYVE, EEA1). Rab5 also recruits the CORVET complex, a fusion machinery allowing fusion with endosomes. The phagosome up until the recruitment Rab7 is called the early phagosome.

Transition to the late phagosome happens when Rab7 is delivered to the phagosome. Quickly Rab5 will be sorted out of the phagosome membrane by the Mon1–Ccz1 complex. Once only Rab7 is detectable the phagosome is known as the late phagosome. Transition to the phagolysosome occurs when the acidic lysosomes fuse with the late phagosome. RIPL, a Rab7 adaptor, anchors the late phagosomes to microtubules, driving the late phagosomes towards the Microtubule Organizing Center. The lysosomes, also anchored to the microtubules and numerous near the Microtubule Organizing Center, fuse with the phagosome. Fusion is detectable by the presence of LAMP1/2 on the membrane of the phagosome. The phagosome is now an acidic Rab7-LAMP1/2 positive phagolysosome.

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In D. discoideum, phagosome maturation follows the “kiss and run” model but unlike in human phagocytes, fusion events are more rapidly orchestrated by Rab7 (Fig.8). Within 3 minutes after ingestion, Rab7 is localized at the phagosome (Rupper et al., 2001) whereas in macrophages it takes 30min (Seto et al., 2011). Early recruitment of the VPS-34 homolog DdPIK5 is also necessary (Zhou et al., 1995) and production of PI(3)P by VPS-34 recruits proteins with PX or FYVE domains such as PIKfyve (Buckley et al., 2019). Fusion with lysosomes is also important for proper maturation in D. discoideum. Similarly to human phagocytes, this fusion is mediated by SNAREs, in this case Vamp7 and syntaxin 7 (Flannagan et al., 2012).The fusion process is at least partially under LvsB control. LvsB inhibits Rab14 which promotes fusion between lysosomes and postlysosomes (Kypri et al., 2013). The main difference between the D. discoideum and human late phagocytic pathway is that the D. discoideum phago-lysosome typically matures into a postlysosome which is similar to the secretory lysosomes of some specialized mammalian cells (Blott et al., 2002). Vacuolar-ATPase, responsible for acidifying the phagosomes, is removed from the membrane of phagosomes in budding vesicles 30 minutes after ingestion (Carnell et al., 2011). The WASH complex, a regulator of actin polymerization is essential for the sorting out of V-ATPase (Carnell et al., 2011). Interestingly in human, WASH is enriched in the early recycling pathway compared to the late degradative pathway, as seen by its preferential colocalization with Rab5 but far less or not with Rab7 and LAMP (Derivery et al., 2019). The postlysosome, devoid of V-ATPase, exhibiting a near neutral pH, and an actin coat eventually fuses with the plasma membrane (Lima et al., 2012).

Fusion of the postlysosome with the plasma membrane is regulated in a Ca²⁺ dependent manner by mucolipin, a member of the transient receptor potential ion channel family.

Other Rab GTPases play important regulating function in the endocytic pathway. In humans, Rab1 and Rab2 mediate the interactions between the phagosome and the ER, post-Golgi and ERGIC compartments (Gutierrez, 2013). Rab14 is involved in Trans-Golgi Network (TGN) to early endosomes and plasma membrane transport.

Rab22A regulates the Rab5 to Rab7 switch. Rab32 and Rab34 regulates in conjunction with Rab7 fusion of the phagosome with the late endocytic pathway (Seto et al., 2011). Lastly, Rab4 and Rab11 regulates phagosomal recycling (Gutierrez, 2013). In D. discoideum, homologs of Rab1, Rab4, Rab11 and Rab14 have similar functions (Harris and Cardelli, 2002).

The recycling machinery

Proteins delivered to the phagosome during maturation, or originating from the plasma membrane, are recycled in order to allow their use in several cycles of phagocytosis (Fig.9). In human cells, cargoes delivered into early sorting endosomes that are destined to be degraded are segregated from cargoes destined to be recycled.

Cargoes destined for degradation are ubiquitinated and recognized by the ESCRT complexes, which induce the formation of multivesicular bodies (MVBs). MVB formation results from the invagination of a portion of the limiting membrane of an endosome into its own lumen (Piper and Katzmann, 2007). The early endosome then matures into late endosome/lysosome, where its content is degraded when the MVBs fuse with lysosomes. On the other hand, cargoes that need to be recycled are spatially segregated into tubular structures of the sorting endosome (Piper and Katzmann, 2007). There, they are transported back to the surface or to the TGN by a “fast”

recycling dependent on the retromer or retriever complexes, or via a “slow” recycling through the endosomal recycling compartment (ERC) (Maxfield and McGraw, 2004; Simonetti and Cullen, 2018). Both recycling pathways relies on Rab GTPases activity and a family of proteins involved in sorting cargoes: the sorting nexin family (SNXs). SNXs bind PI(3)P, via their PX domain. A subset of SNXs also contains BAR domains and induces membrane bending and tubulation (Worby and Dixon, 2002).

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Fig.8 Phagosome maturation in Dictyostelium discoideum

Once closed, the phagosome containing bacteria sheds its actin coat, and rapidly acquires the V-ATPase and Rab7. The Rab7 positive phagosome fuses rapidly with lysosomes. Rapid acidification of the phagosome luminal pH is necessary for the lysosomal enzymes function. Fusion between phagolysosomes and lysosome is stimulated by Rab14. The phagolysosomes stay at peak acidity during approximately 20-30 min. Quickly after the V-ATPase and lysosomal enzymes are recycled by the WASH complex and the phagosome acquires LvsB, preventing fusion with acidic compartments. The phagolysosomes mature in a pH neutral large vacuolar named postlysosome. 40 to 60 min after, non-digested bacterial components are exocytosed by fusion of the postlysosome with the plasma membrane.

Exocytosis speed is partially under the control of MclN, which store calcium in the postlysosome lumen, slowing down fusion with the plasma membrane.

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Regarding the “fast” recycling, the retromer and retriever complexes are both heterotrimers composed respectively of: Vps35, Vps29, Vps26 and DSCR3, C16orf62, Vps29 (McNally et al., 2017, Gallon and Cullen, 2015). Regulation of how Vps29 interacts with the retromer or the retriever complex is unknown. However, both complexes interact with SNXs. The Retromer complex is known to interact with SNX-BAR heterodimers and ensures the recycling of membrane proteins from early endosomes to the TGN (Gallon and Cullen, 2015).

The retromer complex can also interact with SNX27, for the recycling of certain plasma membrane proteins (Seaman, 2012; Burd and Cullen, 2014; Gallon and Cullen, 2015). Regarding the retriever complex, it was proposed to interact with other SNXs, such as SNX17, which binds NPx(Y/F)/Nxx(Y/F) motifs present on certain plasma membrane proteins, for example β1-integrin (McNally et al., 2017; McNally and Cullen, 2018).

An important partner of both the retromer and retriever is the WASH complex, an NPF responsible for activating the Arp2/3 complex, and thus driving actin polymerization (Carnell et al., 2011). The WASH complex localizes at retromer-enriched membranes on the membrane of early endosomes. Interaction with the retromer is carried out by the binding of the WASH complex subunit FAM21 with Vps35 (McGough et al., 2014). The WASH/Retromer complex creates an emerging tubule from the endosome, further pulled by dynein bound to SNXs. A scission mediated by EHD1 and dynamin separates the vesicle from the endosome (Lucas et al., 2016).

EHD1 is one of the four members of the C-terminal Eps15 homology domain family of proteins which are ATPases that bind endocytic membranes and induce tubulation or vesiculation (Naslavsky and Caplan, 2011).

WASH activity in early endosomes allows fast recycling of surface proteins back to the plasma membrane and retrograde transport of phagosome content to the TGN (Derivery et al., 2009; Gomez and Billadeau, 2009).

Regarding the “slow” recycling, the ERC, found at the juxtanuclear region, is enriched in Rab11 which is necessary for transport of proteins to the plasma membrane (Maxfield and McGraw, 2004). The ERC function is also regulated by SNX4 and members of the EHD family (Traer et al., 2007, Grant and Donaldson, 2009;

Naslavsky and Caplan, 2011). SNX4 plays an important role in sorting the transferrin receptor (TfR) from the sorting endosome to the ERC (Traer et al., 2007) and EHD1 and EHD4 are involved in vesiculating tubular structures of the ER allowing transport of cargoes to the plasma membrane (Cai et al., 2013).

In D. discoideum, like in human phagocytes, plasma membrane components are efficiently recycled from an early endocytic compartment (Neuhaus and Soldati, 2000, Vines and King, 2019). Two phases can be distinguished. A fast phase, mediated by the WASH and retromer complexes, in an myoB-independent manner.

A slow phase, relying on a juxtanuclear recycling compartment regulated by Rab11b, vacuolin and potentially MyoB activity (Neuhaus and Soldati, 2000, Bosmani et al., 2018). WASH participates in recycling of certain plasma membrane proteins, including SibA, presumably through its interaction with the retromer complex (Buckley et al., 2016). WASH induces F-actin polymerization providing forces to pull tubules from the sorting endosome (Simonetti and Cullen, 2018). Unlike in human phagocytes, the interplay between dynamin and EHD for the scission of the tubule is poorly understood. The unique EHD protein in D. discoideum induces vesiculation and scission of endosomal tubules, like DymA the dynamin ortholog in D. discoideum, but is delivered to the phagosome independently from DymA (Gueho et al., 2016). Moreover in D. discoideum, WASH, EHD and DymA overlaps only for a few minutes on phagosomes in the early stages and association between the WASH complex and EHD/DymA has not been demonstrated yet (Gueho et al., 2016).

While WASH promotes actin polymerization on the phagosome and retrieval of cargoes, the actin network coating the phagosome also prevents efficient delivery (Dieckmann et al., 2012). Abp1, a major F-actin binding protein on phagosomes, play a critical role: in abp1 KO cells lysosomal fusion to phagosomes is increased, likely due to a disorganized actin network on the phagosome (Dieckmann et al., 2012). Abp1 regulation is important for the function of DymA in the early maturation step and MyoB in the late stage (Gopaldass et al.,

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2012). Proper delivery and retrieval of material back and forth from the phagosome require a tight regulation of the phagosome actin coating.

Fig.9 The recycling machinery

In mammalian cells, cargoes delivered for degradation into the early endosome are segregated from cargoes destined for recycling. Cargoes for degradation will be ubiquitinated and recognized by the ESCRT complex. It induces formation of multivesicular bodies (MVBs). Early endosomes then mature into late endosomes, where their content is degraded when the MVBs fuse with lysosomes.

On the other hand, cargoes that need to be recycled are spatially segregated into tubular structures of the sorting endosome. They are transported back to the surface or to the TGN by a “fast” recycling dependent on the retromer or retriever complexes, or via a “slow” recycling through the endosomal recycling compartment (ERC). Both recycling pathways relies on Rab GTPases activity and a family of proteins involved in sorting cargoes: the sorting nexin family (SNXs).

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IV. Intracellular killing mechanism: comparative analysis in human and D. discoideum

While the phagosome matures, a linear sequence of event unfolds starting with the acidification of the lumen of the phagosome, quickly coupled with the production of reactive oxygen species (ROS) and followed by the delivery of lysosomal enzymes. At the same time, metal transporters selectively deplete several metal ions and pump toxic metal ions into the phagosomal lumen (Dunn et al., 2018). All these mechanisms contribute to killing and digesting the phagocytosed bacteria. Role of autophagy will also be discussed in this section.

Acidification of the phagosome lumen

Acidification of the phagosome lumen by the H⁺ V-type (vacuolar) ATPases minutes after closure was first measured 30 years ago (Lukacs et al., 1990). This proton pump consists of two subcomplexes, the membrane associated V0 and cytosolic V1 complexes. The V1 subcomplex is responsible for hydrolysis of the ATP, which causes a conformational change allowing the V0 complex to pump H⁺ protons inside the phagosome lumen (Maxson and Grinstein, 2014). The V-ATPase is delivered by fusion to the phagosome of lysosomal vesicles or tubules (Sun-Wada et al., 2009). The lowest pH reached between 10 and 30 min after phagosome formation in macrophages is 4,5–5 (Sun-Wada et al., 2009). Acidification plays an important role in both intracellular killing and phagosome maturation: knocking down a subunit of the V-ATPase, leads to a pH neutral phagosome.

It does not completely prevent maturation of the phagosomes but prevents efficient bacterial killing (Sun-Wada et al., 2009). pH alone can adversely affect bacterial growth, for E. coli growth is halted in media at pH 4 (Tsuji et al., 1982) and killed below pH 2.5 (Zhu et al., 2006) but in the phagosomes the pH does not drop below 4.5.

It is more likely that acidification is required for several other IC killing mechanisms to take place (Sattler et al., 2013) (Fig.10).

Pathogenicity of several microorganisms relies on hijacking the V-ATPase function: either by preventing its delivery to the phagosome or inhibiting its function. In the case of M. tuberculosis, the pathogen produces PtpA, a protein which binds a subunit of the V-ATPase blocking its insertion in the phagosome membrane (Wong et al., 2011). Numerous others bacteria such as, H. capsulatum, Streptococcus pyogenes, Rhodococcus equi, Yersenia pestis, and even fungi such as C. albicans prevent the insertion of the V-ATPase in the phagosome membrane (Strasser et al., 1999, Nordenfelt et al., 2012, Toyooka et al., 2005, Pujol et al., 2009, Fernández- Arenas et al., 2009). Failure to hijack the V-ATPase function or delivery often results in less pathogenic strains (Uribe-Querol and Rosales, 2017).

Optimizing H⁺ accumulation requires also efflux of cations and influx of anions, such as chloride through CFTR and CLC transporters (Di et al., 2006, Soldati and Neyrolles, 2012), to dissipate the development of a restrictive electrical gradient induced by the H+ influx (Di et al., 2017). Furthermore, it appears that chloride transporter plays an additional role in regulating the release of luminal Ca²⁺, which is essential for phagosome-lysosome fusion in macrophages (Wong et al., 2017). Interestingly, a pH and voltage dependent selective proton channel Hv1 is enchased in the phagosome membrane. Hv1 allows a more pronounced acidification of the phagosome in macrophages at the cost of lower ROS production. On the contrary, it maintains a neutral phagosomal pH to sustain high ROS production in neutrophils (El Chemaly et al., 2014). This dichotomy is due to the ability of Hv1 proton channels to prevent the luminal alkalinisation caused by ROS production, which in neutrophils results in lower accumulation of VATPases that would acidify phagosomes (El Chemaly et al., 2014).

In D. discoideum, V-ATPase is rapidly delivered to the phagosomes and they acidify faster (Clarke et al., 2002) (Fig.11). In D. discoideum a GFP-tagged subunit of the V-ATPase is delivered to yeast-containing phagosomes 1-2 minutes after their closure and maximum acidification is reached 15 min after ingestion (Clarke et al., 2002).

In mouse macrophages, the same delivery takes 6 min and the phagosome reaches its lowest pH 60 min after

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INTRODUCTION

ingestion (Sun-Wada et al., 2009). This difference is most likely explained by the need for D. discoideum to ensure a rapid action of its lysosomal enzyme that requires an acidic pH (Sattler et al., 2013). D. discoideum phagosomes are also more acidic than mouse macrophages, with pH 4 or below (Marchetti et al., 2009, Sattler et al., 2013). Achieving a lower pH for D. discoideum ensures growth inhibition for a wider range of bacteria.

As previously described, in D. discoideum the V-ATPase is sorted out of the postlysosome membrane 45-60min after ingestion by the WASH complex. Unfortunately, less studies have identified and characterized the counter- ion channels in D. discoideum on the phagosome membrane, the closest homolog to the chloride channel CFTR is ABCC.8 a member of ABC superfamily of transporter (Anjard et al., 2002) but has not been characterized, nor homologs to the cation transporter TRPM2 or even Hv1.

To survive in the highly acidic phagosome of D. discoideum, pathogens use similar strategies to those characterized in macrophages. For example, infection by M. marinum of D. discoideum, is very similarly to M.

avium infection in macrophages. The mycobacterium initially prevents delivery or promotes extraction of the V-ATPase from the phagosome membrane. In D. discoideum, barely 50% of the phagosomes display the presence of a GFP-tagged subunit of the V-ATPase in the M. marinum containing phagosomes 20 min post infection; and the number keeps decreasing with time (Hagedorn et al., 2007).

Creating a proton gradient ensures not only regulation of the pH, in both organisms, but also transport of other ions in or out of the phagosome as well as transport and optimal activity of lysosomal enzymes, which will be subsequently described. Thus, association and dissociation of the v-ATPase is highly regulated during phagocytosis (Maxson and Grinstein, 2014).

ROS production in the phagosome

Enzymes that scavenge superoxide (O2−) and hydrogen peroxide (H2O2), such as superoxide dismutase or peroxiredoxins or catalases, are ubiquitously expressed in eukaryotic organisms. This observation drove research in oxidative stress since McCord in 1971 hypothesized that oxygen toxicity is mediated by partially reduced oxygen species, which are by-products of the aerobic metabolism and should be kept in check (McCord et al., 1971). Due to the high reactivity of the partially reduced oxygen species, they were labelled reactive oxygen species (ROS). Since their discovery ROS have been shown to function as antimicrobial effectors (Kovacs et al., 2015), signaling molecules that regulate NF-κB, (Yang et al., 2013), autophagy (Huang et al., 2009), cytokine secretion (Liu et al., 2014), inflammasome activation (Cruz et al., 2007), apoptosis (Miller et al., 2010), cytoskeleton dynamics and chemotaxis (Stanley et al., 2014). Generation of ROS is carried out by members of the NADPH oxidase (NOX) family (Buvelot et al., 2019). More specifically, the production of ROS in the phagosome of neutrophils and macrophages specifically has been linked to the NADPH oxidase:

NOX2 (Royer-Pokora et al., 1986). NOX2 generates superoxide by transferring electrons from the cytosolic NADPH to a luminal O2 molecule. The phagocyte oxidase (phox) complex comprises five subunits:

gp91^phox/Nox2, p22^phox, p40^phox, p47^phox, and p67^phox. The phox complex also requires Rac1 and 2 for complete activation (Abo et al., 1991, Knauss et al., 1991). Two main enzymes subsequently convert superoxide into other ROS: superoxide dismutase and myeloperoxidase. The superoxide dismutase catalyzes superoxide conversion to hydrogen peroxide (H2O2). The myeloperoxidase, which catalyzes the production of hypochlorous acid HOCL (Nguyen et al., 2017). Hypochlorous acid (HOCl) is highly bactericidal at neutral or low pH (Levine and Segal, 2016). H2O2 oxidizes ferrous iron to generate highly reactive hydroxyl radicals •OH through a mechanism known as the Fenton reaction. Protons and chloride are provided for these reactions respectively by Hv1 and chloride transporters of the CFTR and CLC family (El Chemaly et al., 2014, Dunn et al., 2018) (Fig.10).